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CD19 targeted chimeric antigen receptor, method of dual-modifying same, and application of the CD19 targeted chimeric antigen receptor

A receptor, single-chain antibody technology, applied in the field of chimeric antigen receptors targeting CD19, which can solve the problems of exceeding the therapeutic requirements, T cell attack, and high expansion volume

Active Publication Date: 2018-07-27
HRAIN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this treatment has not been perfected, and T cells will go off target and attack other tissues, or expand too much, beyond the treatment needs

Method used

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  • CD19 targeted chimeric antigen receptor, method of dual-modifying same, and application of the CD19 targeted chimeric antigen receptor
  • CD19 targeted chimeric antigen receptor, method of dual-modifying same, and application of the CD19 targeted chimeric antigen receptor
  • CD19 targeted chimeric antigen receptor, method of dual-modifying same, and application of the CD19 targeted chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1: Determination of CD19scFv-CD8α-CD28-CD3ζ-tEGFR-aPD1scFv gene sequence

[0082] 1. From the NCBI website database searched anti-CD19 antibody light chain and heavy chain variable regions, human CD8α hinge region, human CD28 transmembrane region and intracellular region, human CD3ζ intracellular region, anti-PD1 antibody heavy chain and The light chain variable region gene sequence information, these sequences are codon-optimized on the website http: / / sg.idtdna.com / CodonOpt to ensure that the encoded amino acid sequence is more suitable for expression in human cells. The whole gene synthesis of the above sequence was introduced, and different enzyme cutting sites were introduced at the beginning and end to form the complete CD19-28z-tEGFR-aPD1 gene sequence information.

[0083] 2. Recombinant plasmid sequencing

[0084] Send the recombinant plasmid to Shanghai Sangon Biotechnology Co., Ltd. for sequencing, and compare the sequencing results with the simulated...

Embodiment 2

[0088] Example 2: Construction of a viral vector comprising the CD19-28z-tEGFR-aPD1 nucleic acid sequence

[0089] The CD19-28z-tEGFR-aPD1 nucleotide sequence prepared in Example 1 was double digested with NotI (NEB) and EcoRI (NEB), connected with T4 ligase (NEB) and inserted into the NotI-EcoRI of the retroviral MSCV vector The site was transformed into competent E.coli (DH5α). After the sequencing was correct, the plasmid was extracted and purified using Qiagen’s plasmid purification kit, and the purified plasmid was transfected into 293T cells by the plasmid calcium phosphate method for retrovirus packaging experiments.

[0090] The plasmid map constructed in this example is as follows figure 1 shown. figure 2 The peak diagram of partial sequencing results of the retroviral expression plasmid is shown.

Embodiment 3

[0091] Example 3: Retroviral packaging

[0092] 1. On the first day, the 293T cells should be less than 20 passages and not overgrown. Plate at 0.6*10^6 cells / ml, add 10ml of DMEM medium to a 10cm dish, mix the cells well, and culture overnight at 37 degrees.

[0093] 2. On the second day, the confluence of 293T cells reached about 90% for transfection (usually about 14-18 hours after plating); prepare plasmid complexes, and the amount of various plasmids is RV-CD19-28z-tEGFR-aPD1 (MSCV backbone plasmid ) is 12.5ug, Gag-pol is 10ug, VSVg is 6.25ug, CaCl 2 250ul,H 2 O is 1ml and the total volume is 1.25ml; add HBS equal to the volume of the plasmid complex in another tube, and vortex for 20 seconds while adding the plasmid complex. Gently add the mixture to the 293T dish along the side, incubate at 37°C for 4 hours, remove the medium, wash it with PBS, and add pre-warmed fresh medium again.

[0094] 3. Day 4: 48 hours after transfection, collect the supernatant and filter ...

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Abstract

The invention relates to a CD19 targeted chimeric antigen receptor and an application thereof, and particularly provides a polynucleotide sequence, which is selected from: (1) a polynucleotide sequence which contains, in a successively connected manner, an encoding sequence of anti-CD19 single-chain antibody, an encoding sequence of human CD8[alpha] hinge zone, an encoding sequence of human CD28 transmembrane zone, an encoding sequence of human CD28 intracellular zone, an encoding sequence of human CD3 [zeta] intracellular zone, and optionally, a fraction, which contains an extracellular domain III and an extracellular domain IV of EGFR, and an encoding sequence of anti-human PD1 sequence fraction; and (2) a complementary sequence of the polynucleotide sequence (1). The invention also provides related fusion proteins, a carrier containing the encoding sequences, and applications of the fusion proteins, encoding sequences and carrier. The CAR-T cell has strong killing effect on specifictumor cells, and can reach more than 90% in killing efficiency on the specific tumor cells under the multiplicity of infection of 1:2. The CAR-T cell can secrete the PD1 antibody and has regulation effect on immunosuppression micro-environments.

Description

technical field [0001] The invention belongs to the field of chimeric antigen receptors, and in particular relates to CD19-targeting chimeric antigen receptors and uses thereof. Background technique [0002] Chimeric Antigen Receptor-T cell (CAR-T) T cells refer to T cells that can recognize specific target antigens in an unrestricted manner by MHC after genetic modification, and continuously activate and expand T cells. In 2012, the annual meeting of the International Society for Cell Therapy pointed out that biological immune cell therapy has become the fourth means of tumor treatment besides surgery, radiotherapy, and chemotherapy, and will become a must-have means for future tumor treatment. CAR-T cell reinfusion therapy is the most clear and effective form of immunotherapy in current tumor treatment. A large number of studies have shown that CAR-T cells can effectively recognize tumor antigens, induce specific anti-tumor immune responses, and significantly improve the ...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N7/01A61K35/17A61P35/00A61P35/02
CPCC07K14/705C07K14/7051C07K14/70517C07K14/70521C07K14/71C07K16/2896C07K2319/00C07K2319/03C12N7/00C12N2740/10021
Inventor 黄飞金涛王海鹰何凤史子啸
Owner HRAIN BIOTECHNOLOGY CO LTD
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